While the essential nucleic acid chemistry of the integration reaction is reasonably understood, how integration of the viral pre-integration complex actually happens in the complex environment of the cell nucleus remains mysterious in many respects. In particular, the integration field is intensely interested in how cellular molecules participate either positively or negatively in the reaction between the pre-integration complex and chromatin, and also in how they determine where and when the reaction occurs in the host cell genome. The transcriptional coactivator LEDGF/p75 has now been securely implicated as an integration cofactor specific for lentiviruses. A basic molecular tethering model has been put forth in which LEDGF/p75 tethers IN to chromatin, key participating protein domains have been identified, and precise structural information exists for the LEDGF/p75 domain that interacts with IN. Hypothesized roles of LEDGF/p75 include acting as a cofactor for catalysis steps, protecting IN and the pre- integration complex in which it resides from degradation, and guiding or tethering the pre-integration complex to chromatin. The latter mechanism could be crucial for the virus to engage chromatin and integrate per se, and it also may determine the distinctive genomic distributions now evident for HIV integration sites. Understanding the importance of LEDGF/p75 to HIV biology may therefore have long-term implications for understanding latency and may provide a new therapeutic target. It could even pave the way for targeting of lentiviral vectors to reduce insertional mutagenesis risks. In this project, we will use effective RNAi knockdown methods, genetic knockout cells, virological analyses, domain mapping and heterologous tethering techniques, genome- wide integration site analyses, and cell biological and proteomic methods to both further understand and exploit the viral biology of LEDGF/p75. PUBLIC HEALTH RELEVANCE This project will elucidate the significance of the transcriptional cofactor LEDGF/p75 in the HIV-1 life cycle, using virological, biochemical, genomic and chimeric protein approaches.